Display of a Functional Nonviral Polypeptide on a Virus which can Infect Eukaryotic Cells
Recombinant viruses have been widely used as vectors for the delivery of foreign genes into eukaryotic cells. Recombinant viruses which are used for delivery of foreign genes to animal cells include members of several virus families, including Adenovirus, Herpesvirus, Togavirus, and Retrovirus families. Viruses which infect eukaryotic cells comprise a protein shell or shells (the capsid) formed by the multimeric assembly of multiple copies of one or more virus-encoded proteins. The capsid houses the viral nucleic acid (RNA or DNA) and may or may not be enveloped in a lipid bilayer which is studded with virus-encoded oligomeric spike glycoproteins, visible on electron micrographs as spikes projecting from the surface of the virus.
The initial event in the virus life cycle is binding to the surface of the eukaryotic target cell. Binding is mediated by the direct interaction of specialised proteins or glycoproteins on the surface of the virus (antireceptors) with receptors on the surface of the target cell, or indirectly via soluble ligands which bind the virus to receptors on the surface of the target cell. In some instances, the interaction between a virus and a target cell receptor may transmit a metabolic signal to the interior of the target cell. Binding is followed by penetration of the target cell membrane and entry of the viral nucleic acid into the cytosol (reviewed in Marsh and Helenius 1989 Adv Virus Res 36 p107-151). Some nonenveloped viruses undergo conformational changes which result in their direct translocation across the target cell membrane, whereas others, such as adenovirus, are first endocytosed and then cause disruption of the wall of the acidified endosomal vesicle. Enveloped viruses fuse with the target cell plasma membrane whereupon the virus capsid (or core particle), housing the viral nucleic acid is released into the cytoplasm of the target cell. This envelope fusion event is catalysed by oligomeric viral membrane spike glycoproteins which are anchored in the viral envelope and may, or may not be dependent on the prior endocytosis of bound virus and its exposure to an acidic environment within the endosomal vesicle. The mechanisms by which viral spike glycoproteins catalyse membrane fusion may involve their proteolytic cleavage, the dissociation of noncovalently linked subunits or other conformational alterations which expose buried hydrophobic moieties capable of penetrating the lipid membrane of the target cell. Thus, virus-mediated delivery of nucleic acid is a complex, multistage process.
After delivery of the viral nucleic acid into the target cell, further steps in the viral life cycle which lead to viral gene expression, genome replication and the production of progeny viruses are often critically dependent on variable host cell factors. For example, division of the infected target cell is required for efficient integration of a reverse-transcribed retroviral genome into the host cell chromosome and subsequent retroviral gene expression (Harel et al, 1981 Virology 110 p202-207).
The spike glycoproteins of one virus can be incorporated into the viral particles of another strain. Thus with dual viral infection of a single cell by two enveloped mammalian viruses, the host range of either virus may be predictably extended due to promiscuous incorporation of spike glycoproteins encoded by both viruses. This has been shown for closely or distantly related retroviruses (Levy, 1977 Virology 77 p811-825; Weiss and Wong, 1977 Virology 76 p826-834; Besmer and Baltimore, 1977 J Virol 21 p965-973; Canivet et al, 1990 Virology 178 p543-541; Lusso et al, 1990 Science 247 p848-852; Spector et al, 1990 J Virol 64 p2298-2308) and for unrelated viruses from different families (Schnitzer et al, 1977 J Virol 23 p449-454; Metsikko and Garoff, 1989 J Virol 63 p5111-5118; Schubert et al, 1992 J Virol 66 p1579-1589). The spike glycoproteins of certain closely or distantly related retroviruses have also proved to be entirely interchangable, allowing production of infectious hybrid virions with envelope spike glycoproteins of one retrovirus and core particles of another retrovirus (Mann et al, 1983 Cell 33 p153-159; Wilson et al, 1989 J Virol 63 p2374-2378). Similar results have been demonstrated with insect/plant viruses (Briddon et al, 1990 Virology 177 p85-94). Furthermore virus host range was predictably altered by exchanging the N-terminal receptor binding domains of envelope spike glycoproteins from related retroviruses with distinct cellular tropisms (Battini et al, J Virol 1992:66 p1468-1475). When viral spike glycoproteins from closely related virus strans were coexpressed in the same cell, mixed oligomers were formed with high efficiency (Boulay et al, 1988 3 Cell Biol 106 p629-639; Doms et al, 1990 J Virol 64 p3537-3540). Thus, there is considerable scope for altering the host ranges of recombinant viruses by exchanging, mixing and recombining the viral spike glycoproteins of naturally occurring viruses.
However, a relatively small minority of the universe of eukaryotic cell surface structures are actually used as receptors by naturally occurring viruses and it is often difficult to identify a virus with a host range which coincides with the requirements of a particular gene therapy application. For example, to genetically modify a population of normally quiescent haemopoietic stem cells in vivo, a most desirable gene transfer vehicle would be a recombinant retrovirus whose spike glycoproteins do not bind to nontarget cells, but which bind to haemopoietic stem cells and mediate membrane fusion and which also signal the target cell to divide as the nucleic acid is delivered. There are no known naturally occurring viral spike glycoproteins which meet these requirements, and there is therefore a need for new technologies to facilitate the generation of novel spike glycoproteins which can enhance the specificity and efficiency of virus-mediated gene delivery and expression.
Preformed viral particles can be attached to cells which lack virus receptors by way of a (multivalent) molecular bridge. This is clearly demonstrated by the phenomenon of antibody-dependent enhancement of viral infectivity. Thus, antibody-complexed foot-and-mouth disease virus (a nonenveloped picornavirus) has been shown to infect normally insusceptible cells via the Fc receptor (Mason et al, 1993 Virology 192 p568-577). The phenomenon of antibody-dependent enhancement of viral infectivity, mediated through binding of antibody-complexed viruses to cellular Fc receptors and complement receptors has been demonstrated for several enveloped and nonenveloped viruses (Porterfield, 1986 Adv Virus Res 31 p335-355). Moreover, bivalent antibodies that bind dengue virus to cell surface components other than the Fc receptor were recently shown to enhance infection (Mady et al, 1991 J Immunol 147 p3139-3144). Also, Baird et al (1990 Nature 348 p344-346) showed that herpes simplex virion penetration into vascular cells via the basic fibroblast growth factor (FGF) receptor requires the association of soluble FGF with the viral particles.
Goud et al, (1988 Virology 163 p251-254) incubated ecotropic murine leukaemia viruses (MLVs) with monoclonal antibodies against the gp70SU viral envelope spike glycoprotein and incubated human HEp2 cells with monoclonal antibodies against the transferrin receptor. Crosslinking of the bound monoclonal antibodies with a sheep anti-mouse kappa light chain antibody allowed the binding of virus on HEp2 cells and its subsequent internalisation into the cells at 37.degree. C. However, internalisation of the virus by this route was not followed by establishment of the proviral state.
Subsequently, Roux et al (1989 Proc Natl Acad Sci USA 86 p9079-9083) and Etienne-Julan et al (1992 J Gen Virol 73 p3251-3255)) used a similar approach in which biotinylated antibodies against the murine ecotropic retroviral envelope spike glycoprotein and against specific membrane markers expressed on human cells were bridged by streptavidin and used to link the virus to the human host cell. The method was successfully used to infect human cells with ecotropic murine retroviruses bound to MHC class I and class II antigens, and to the receptors for epidermal growth factor and insulin. However, targeting of the transferrin, high density lipoprotein and galactose receptors, and of various membrane glycoconjugates, by murine ecotropic retroviruses did not lead to the establishment of a proviral state.
Preformed viral particles can also be chemically modified to facilitate their binding to target cells which lack receptors for the unmodified virus (Neda et al, 1991 J Biol Chem 266 p14143-14149). Murine ecotropic retroviral particles which had been chemically modified with lactose were shown to bind specifically to the asialoglycoprotein receptor on human HepG2 cells (which lack receptors for murine ecotropic viruses). Binding was followed by retroviral infection of the human cells as indicated by transfer of a functional .beta.-galactosidase gene.
Thus, the display of a functional non-viral polypeptide at the surface of the virus can lead to the preferential binding of the modified viral particles to selected target cells, and in some cases, dependent on the exact specificity of the displayed polypeptide, binding is followed by delivery and expression of the encapsidated viral nucleic acid. However, the present inventors realised that a method for the production of viral particles which incorporate and display a nonviral polypeptide during their assembly would be more useful, avoiding the need for modification of preformed virions Such a method would also facilitate the use of such recombinant viruses as genetic display packages (see below).
Non-viral proteins have been incorporated, during assembly, into viral particles capable of infecting eukaryotic cells. Thus, spontaneous incorporation of non-virus-encoded mammalian cellular proteins has been observed in retroviral particles. For example, MHC antigens are incorporated into the envelopes of human and simian immunodeficiency viruses (Gelderblom et al, 1987 Z Naturforsch 42 p1328-1:334; Schols et al, 1992 Virology 189 p374-376). Also, mammalian CD4 expressed in avian (quail) cells was incorporated into the envelopes of budding avian retroviruses (Young et al, 1990 Science 250 p1421-1423). Viral incorporation and display of CD4 was also demonstrated in recombinant herpes simplex virions constructed by inserting the CD4 gene into the HSV-1 genome under the control of a viral promoter (Dolter et al, 1993 J Virol 67 p189-195). However, nonviral proteins are generally excluded from assembling viral particles, or incorporated very inefficiently. The present inventors believe a more reproducible strategy for the efficient incorporation and display of nonviral polypeptides on viruses which can infect eukaryotic cells would be to fuse the nonviral polypeptide to a viral component, such as a viral spike glycoprotein, which carries a signal for incorporation into the viral particle.
The nucleic acid sequences encoding a nonviral peptide or polypeptide can be linked, without disruption of the translational reading frame, to nucleic acid sequences coding for all or part of the gene III protein of filamentous bacteriophage, thereby creating a hybrid gene encoding a chimaeric gene III protein (McCafferty et al, 1990 Nature 348 p552-554; Smith, 1985 Science 228 p1315-1317; Parmley and Smith, 1988 Gene 73 p305-318; Scott and Smith, 1990 Science 249 p404-406). The details of the construction can be varied such that the gene III moiety of the chimaeric protein remains substantially intact or is lacking one or more domains. When expressed in prokaryotic cells which are shedding filamentous bacteriophage, the chimaeric gene, III protein is incorporated into a proportion of the progeny phage and those phage display the chimaeric protein on their surface. Incorporation of the chimaeric protein is presumed to occur during phage assembly through the specific interaction of the C-terminal gene III moiety of the chimaeric protein with other protein components of the phage particle. Correct folding and function of the nonviral polypeptide moiety of the incorporated chimaeric gene III protein is apparent from the altered binding specificity of the phage particles, which corresponds with the binding specificity of correctly folded nonviral polypeptide moiety. It is possible to generate phage particles incorporating a combination of wild type and chimaeric gene III protein or incorporating exclusively the chimaeric gene III protein.
However, bacteriophage do not infect eukaryotic cells and cannot therefore be employed as gene delivery vehicles for such cells. Nor can they be used for the display of glycoproteins since their bacterial hosts lack the necessary enzymatic machinery for correct glycosylation of polypeptides.
In the case of nonenveloped viruses which can infect eukaryotic cells, initial binding to the target cell is mediated by the viral capsid which is composed of a multimeric symmetrical array of virus-encoded capsid proteins. Short (up to 26 aminoacids) nonviral peptides have been displayed on the surface of nonenveloped polioviruses by replacing surface-exposed polypeptide loops (up to 9 aminoacids) in the capsid proteins (Rose and Evans, 1991 Trends Biotech 9 p415-421). This was achieved by genetic manipulation and subsequent transfection of a full-length infectious cDNA clone of the RNA genome. The display of peptides in poliovirus antigen chimaeras has been pursued with a view to using the chimaeric particles as antigen presentation vehicles to stimulate immune responses against the displayed peptide in vaccinated animals or humans and for use as diagnostic reagents in serodiagnosis. Larger, functional, folded polypeptides have not been displayed on the outer surface of nonenveloped viruses which can infect eukaryotic cells.
In the case of all enveloped viruses and certain nonenveloped viruses (eg adenovirus) which can infect eukaryotic cells, initial binding to the target cell is mediated by specialised multifunctional, oligomeric spike glycoproteins rather than by simple unglycosylated monomeric proteins such as the gene III protein of filamentous bacteriophage. Thus, it is not possible simply to extrapolate from the bacteriophage gene III display system in the design of chimaeric variants of these oligomeric spike glycoproteins for incorporation into assembling virus particles and display of functional nonviral polypeptides at the surface of the virion. Correct glycosylation and oligomerisation of the spike glycoproteins of enveloped viruses is often required for successful transport to the cell surface and incorporation into viral particles (Polonoff et al, 1982 J Biol Chem 257 p14023-14028; Enfield and Hunter, 1988 Proc Natl Acad Sci USA 85 p8688-8692: Kreis and Lodish, 1986 Cell 46 p929-937; Copeland et al, 1988 Cell 53 p197-209). Proteolytic cleavage of assembled oligomeric viral spike glycoproteins frequently occurs during their transport through the Golgi apparatus to the cell surface. Thus, trimeric Murine Leukaemia Virus envelope glycoprotein precursors are proteolytically cleaved in the Golgi apparatus into p15TM transmembrane and gp70SU surface components, and these components are held together by noncovalent interactions or by covalent disulphide bonds (Pinter et al, 1978 Virology 91 p345-351).
Non-viral polypeptides have been displayed on a retrovirus by fusion to the membrane anchor sequence of the retroviral spike glycoprotein. Adopting this strategy, incorporation of chimaeric CD4-envelope proteins was demonstrated by immunoprecipitation of purified retroviral (RSV) particles, but there was no evidence for correct folding or function of the CD4 (Young et al, 1990 Science 250p11421-1423). Chimaeric VSV-G proteins comprising the cytoplasmic and transmembrane anchor domains of VSV-G spike glycoprotein fused to the ectodomain of CD4 were incorporated into the envelopes of infectious VSV particles (Schubert et al, 1992 J Virol 66 p1579-1589). However, the authors were not able to demonstrate correct folding or function of the virally incorporated CD4 and state that "Numerous experiments to demonstrate a specific tropism for HIV envelope-expressing cells were not successful so far. In the environment of a viral membrane, the receptor may not be functional".
The technique of insertional mutagenesis has been used to define domains of the MoMLV genome which are amenable to small alterations without deleterious effects on the virus (Lobel and Goff, 1984 Proc Natl Acad Sci USA 81 p4149-4153). Viable linker insertions in the env gene of an infectious molecular clone of MoMLV (eg in-6438-12 and in-7407-9) were shown to generate infectious retroviruses whose spike glycoproteins had presumably incorporated the four-residue nonviral peptide encoded by the inserted linker. However, no attempt was made to demonstrate display of such a peptide, nor was the possibility of surface display mentioned.
The present inventors have devised a novel strategy for the incorporation and display of nonviral polypeptides in fusion with viral glycoproteins, particularly oligomeric viral spike glycoproteins. The nucleic acid sequences encoding a nonviral polypeptide are fused, without disruption of the translational reading frame, to nucleic acid sequences coding for the oligomeric viral spike glycoprotein. The hybrid gene codes for a chimaeric glycoprotein in which the domain structure and organisation of the viral spike glycoprotein moiety remain substantially intact so as to conserve the post-translational processing, oligomerisation, viral incorporation and, possibly, fusogenic activities. The nonviral polypeptide is fused close to the terminus of the mature spike glycoprotein which is known to be displayed distally on the outside of the viral particle. To avoid possible steric hindrance between the nonviral polypeptide moieties which could significantly inhibit oligomerisation, the chimaeric glycoprotein can be expressed in virus-shedding cells in the presence of the wild-type virus spike glycoprotein such that each oligomeric unit need incorporate only a single copy of the chimaeric glycoprotein. Adopting this strategy, we have demonstrated incorporation of a chimaeric glycoprotein comprising a single chain antibody fused to a retroviral spike glycoprotein into murine ecotropic and amphotropic retroviral (MLV) particles. Moreover, in contrast to previous studies we have been able to demonstrate that the virally incorporated single chain antibody remains functional as evidenced by its ability to bind specifically lo its target antigen (NIP).
A logical extension of these studies is the construction of vectors of similar design for the display of nonviral peptides, polypeptides and glycopolypeptides other than single chain Fv antibody fragments on retroviral particles. Among the polypeptides and glycopolypeptides suitable for display on the particles are Fv and Fab antibody fragments, T-cell receptors, cytokines, growth factors, enzymes, cellular adhesion proteins such as integrins and selecting, Fc receptors etc. There is no reason why two or more different nonviral polypeptides should not be incorporated into a single virus particle by their co-expression as fusion proteins in the same packaging cell. The display of nonviral peptides, polypeptides or glycopolypeptides as similar fusions with the oligomeric spike glycoproteins of other retroviruses and with viruses of other families also follows directly from the this invention. Animal viruses of the Adenovirus, Togavirus, Rhabdovirus, Paramyxovirus, Orthomyxovirus and Retrovirus families which have relatively well-defined oligomeric spike glycoproteins are particularly suitable for such manipulation.
The invention, in one aspect, thus provides a recombinant viral particle capable of infecting eukaryotic cells, comprising a non viral polypeptide fused to a substantially intact viral glycoprotein or chimera of viral glycoproteins and a displayed on the external surface of the particle.
The term "viral glycoprotein" means a glycoprotein encoded by a virus in its natural state. The viral glycoprotein is typically a viral spike glycoprotein, i.e. a protein which in its natural state:
1. projects from the surface of the virus to be visible by electron microscope; PA1 2. is oligomeric, having 2 to 6 subunits which may be identical or non identical, i.e. homo or heterooligomers; PA1 3. is glycosylated; PA1 4. comprises a structural signal which directs its efficient incorporation into the viral particle. PA1 (a) had incorporated the fusion polypeptide encoded by the construct comprising the Moloney MLV (mouse ecotropic) envelope spike glycoprotein fused to a hapten-binding single chain antibody; PA1 (b) bound specifically to the hapten recognised by the single chain antibody; PA1 (c) encapsidated the nucleic acid sequences encoding the displayed fusion polypeptide; PA1 (d) delivered the encapsidated nucleic acid to murine target cells, whereupon it was reverse transcribed, integrated and expressed; PA1 (e) did not deliver the encapsidated nucleic acid to human target cells. Moreover, sequences encoding the nonviral polypeptide moiety of the displayed fusion polypeptide were amplified and recovered from target cells which had been infected by the retroviral genetic display packages. Murine amphotropic packaging cell lines expressing the construct were also shown to shed retroviral particles that had incorporated the fusion polypeptide, bound specifically to the hapten NIP and were infectious for mammalian cells. PA1 a) Targeted corrective gene replacement therapy for defects of genes encoding intracellular, cell surface or secreted proteins. For example targeted ex vivo or in vivo delivery of genes to correct the defect in sickle cell anaemia or thalassaemia (globin genes to bone marrow progenitor cells), alpha-1 antitrypsin deficiency (peptides to prevent intracellular accumulation of mutant (Z) alpha-1 antitrypsin), haemophilia (factor VIII or factor IX genes to hepatocytes), familial hypercholesterolaemia LDL receptor to hepatocytes). PA1 b) Intracellular immunisation, for example targeted in vivo delivery (to CD4 expressing cells) of genes encoding proteins, antisense transcripts or ribozymes which interrupt or abort HIV life cycle following virus entry. PA1 c) Pharmacological gene addition, for example delivery of genes encoding therapeutic antibodies, growth factors or cytokines to specific tissues in vivo. PA1 d) Cancer therapy. Delivery of genes encoding proteins which destroy the target cell (for example, a ribosomal toxin), indirectly stimulate destruction of target cell by natural effector cells (for example, strong antigens to stimulate immune system) or convert a precursor substance to a toxic substance which destroys the target cell (for example, a prodrug-activating enzyme). Encoded proteins could also destroy bystander tumour cells (for example with secreted antitumour antibody-ribosomal toxin fusion protein), indirectly stimulate destruction of bystander tumour cells (for example cytokines to stimulate immune system or procoagulant proteins causing local vascular occlusion) or convert a precursor substance to a toxic substance which destroys bystander tumour cells (e.g. enzyme which activates prodrug to diffusible drug). Also, delivery of genes encoding antisense transcripts or ribozymes which interfere with expression of cellular genes critical for tumour persistence (for example against aberrant myc transcripts in Burkitts lymphoma or against bcr-abl transcripts in chronic myeloid leukaemia). PA1 (a) As virus components for applications outlined in 10 and 11 above. PA1 (b) As components of any gene delivery vehicle (e.g. liposome, virosome, directly conjugated to DNA, physically linked to surface of preformed virus). PA1 (c) As therapeutic or diagnostic protein reagents (e.g. tumour-targeting reagent). PA1 (d) As components of such therapeutic or diagnostic reagents. PA1 (e) As a means to clone the genes encoding the cell surface components to which they bind and subsequent use of such cell surface components.
The chimera of viral glycoproteins must be capable of incorporation into the viral particle to satisfy conditions 1 to 4 above. One example of such a chimera is a protein comprising the transmembrane and cytoplasmic domains of the trimeric Rous Sarcoma Virus envelope glycoprotein fused to the trimeric extraviral domains of the influenza haemagglutinin (Dong et al., 1992 J. Virol. 66, 7374-7382).
The virus is conveniently a retrovirus, but this is not critical.
The eukaryotic cells are typically mammalian, e.g. human.
The polypeptide and glycoprotein are fused together, i.e. in the form of a single, continuous polypeptide chain.
It is important that the viral glycoprotein is substantially intact, i.e. retains all its domains, to conserve the post-translational processing, oligomerisation, viral incorporation and, possibly, fusogenic activities. However, certain alterations, e.g. mutations, deletions, additions, can be made to the glycoprotein which do not significantly affect these functions, and glycoproteins with such modifications are considered substantially intact.
The non viral polypeptide is preferably fused close to the terminus of the mature glycoprotein which is known to be displayed distally on the outside of the viral particle, so that folding of the distal terminal domain is not significantly disturbed.
The non viral polypeptide generally comprises at least 6 amino acids, and may range from a short polypeptide to a fully functional protein. The polypeptide may be glycosylated. The polypeptide typically comprises an antibody or antibody fragment, receptor, enzyme etc.
The non viral polypeptide can be selected to bind to a target eukaryotic cell, via a cell surface molecule, with the virus delivering encapsidated nucleic acid to the target cell. The viral particles of the invention thus find application in targeted gene delivery and targeted virotherapy, as will be discussed in more detail below.
The non viral polypeptide conveniently comprises antibody or antibody fragments e.g. heavy and light chain variable domains of an antibody, which may comprise framework regions homologous to the framework regions of human antibodies. Such variable domains are conveniently derived from phage display libraries, e.g. by being selected for binding to cell surface molecules.
Antibody fragments of low immunogenicity and peptides with desirable binding activities may be most versatile targeting agents for incorporation into particles for targeting human cells. Antibody fragments could be expressed as single chain fragments (in which the heavy and light chain variable domains are located on the same polypeptide, using by a flexible peptide linking the two domains directly) or as two chain constructs, in which one chain is fused to the second polypeptide and the other is secreted and associates with the fusion protein. Other possibilities for targeting the virus to the cell surface include cytokines, for example EGF for tumours with high expression, or T-cell receptors cloned from tumour-specific T cells.
For targeted binding, for example to human tumour cells, the virus particles should ideally not bind to other human cells. The non viral polypeptide should therefore confer novel (e.g. antitumour) binding activity on the particle. It is therefore desirable to use viral particles which do not naturally bind to the target cells, or which have been modified to destroy natural binding to the target cells. In this case, all binding to target cells will be attributable to the non viral polypeptide.
The target cell specificity of such binding may be further enhanced where two or more novel (e.g. antitumour) binding activities are displayed on a single particle. The viral particle may thus include one or more additional viral coat proteins, in addition to the fusion protein. Such additional proteins may or may not bind to the target cells and may or may not allow infection of the target cells. Additional binding activities conferred by the non viral polypeptide or viral protein moiety of the fusion protein or by unmodified coat proteins may decrease the specificity of binding to the tumour cells, and it will therefore often be desirable to choose these proteins to give minimal background or to inactivate their binding activity, for example by site directed mutagenesis.
After virus binding, fusion with or translocation across the limiting membrane of the target cell, sometimes preceded by endocytosis, is a necessary step which may require inclusion of specific (e.g. fusogenic) proteins in the coat of the viral particle (and these proteins should ideally not increase the background binding to nontarget cells). For example, inclusion of fusogenic influenza haemagglutinin trimers, mutated to destroy sialic acid receptor binding activity, could trigger low pH-dependent membrane fusion of endocytosed virus with the endosomal membrane. Where the nonviral polypeptide is fused to a viral coat protein, the coat protein moiety may itself carry the necessary fusogenic or translocating capability.